Cellular immunity is mediated largely through T-cells which interact with foreign antigens presented in the context of classical MHC class I, class II and non-classical MHC molecules. The main goal of this proposal is to understand the structural basis for T-cell recognition and T-cell signaling. The long term goal is to assemble a complete TCR signaling complex that includes the alpha/betaTCR, pMHC,CD3gamma, delta, epsilon, zeta and CD8. T-cell development will be probed through structural studies of the pre-T-cell receptor. A comparison of how gamma/delta TCRs recognize their antigens compared to alpha/betaTCRs will be evaluated from the gamma/deltaTCR/T10 or T22 nonclassical MHC complex. The alpha/beta, gamma/delta and pre-T-cell receptors all recognize and interact with CD3, but their interactions differ as the components of the various TCR types are not conserved. The CD3 components will be studied independently and as complexes with the different TCRs. In addition, the CD8 co-receptor interaction will be investigated as TCR-pMHC-CD8 complex. Many key questions will be addressed including the structural principles of TCR/MHC recognition that define MHC restriction, high affinity TCR binding is accomplished, whether conformational flexibility (entropic) is reduced in the CDR's of these high affinity TCRs, whether CD8 interacts with both pMHC and TCR, and the major question concerning the structure, stoichiometry and function of the T-cell signaling complex. This later goal is ambitious but represents the only way to probe the structural basis of T-cell signaling. The benefits from this work include an increased understanding of the immune response to microbial pathogens and elucidation of the structural basis of tolerance, autoimmunity, alloreactivity, cross-reactivity and host versus graft disease in transplantation. The IRPG represents a joint effect by two laboratories (Dr. lan Wilson, project I - structural immunology and Dr. Luc Teyton, project II - cellular immunology) to clone, express and purify sufficient quantities of alpha/betaTCR, pre-TCR, pMHC, CD8 (alpha/alpha, alpha/beta) and CD3gamma, delta, epsilon, in order to characterize biochemically and immunologically these TCR components, to crystallize each component alone and in complex with their relevant ligands and to determine their X-ray structures. The synergy between these proposals tremendously enhances and complements each individual project and substantially enhances the likely success of each.